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[Bio101] Purification of Adenovirus by Cesium Chloride Density Gradients
[Bio101] 通过氯化铯密度梯度纯化腺病毒   

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Abstract

Adenovirus are efficient gene delivery systems. The standard method for purification of adenoviral vectors is based on using a cesium chloride (CsCl) density gradient combined with ultracentrifugation. This method is suitable for small-scale purification and is less expensive than column chromatography or commercial purification kits.

Materials and Reagents

  1. HEK293 cell
  2. Glycerol
  3. Liquid nitrogen
  4. 70% ethanol
  5. Distilled water
  6. SDS
  7. CaCl2
  8. MgCl2
  9. CsCl2 (Thermo Fisher Scientific, catalog number: BP1595-500 )
  10. Tris-HCl (pH 8.0)
  11. TE
  12. EDTA
  13. SDS
  14. Sucrose
  15. 5% deoxycholate (see Recipes)
  16. Dialysis buffer (see Recipes)
  17. Balance buffer (see Recipes)
  18. Saturated CsCl2 (see Recipes)

Equipment

  1. Centrifuges
  2. Ultracentrifuge
  3. Laminar flow hood
  4. Syringe
  5. Water bath
  6. Slide-A-Lyzer 10 K dialysis cassette (Thermo Fisher Scientific, catalog number: 66203 )
  7. Spectrometer
  8. Ti50.2 tube

Procedure

  1. Thaw HEK293 cell suspension (with adenovirus in TE +5% glycerol 20.5 ml) from liquid nitrogen at 37 °C.
  2. Add 1.23 ml 5% deoxycholate to 0.3% final concentration and vortex.
  3. Incubate on ice for 30 min (vortex every 10 min). During incubation, prepare homoginizer: wash with 70% ethanol, then with distilled water, then sterilize under ultraviolet light.
  4. Homoginize until cell suspension becomes free flowing (set to 25, three times, 2 min for homogenization and 3 min on ice each time).
    Wash homogenizer: 1x 1% SDS, 5x regular water, 3x sterilized dH2O, dry up.
  5. Incubate cell suspension on ice for 15 min (vortex each 5 min).
  6. Spin 30 min at 9,000 x g to remove cell debris on 4 °C.
  7. Load 11.9 ml of saturated CaCl2 and 20.5 ml of the homogenate onto an ultracentrifuge tube (fit to Ti50.2 tube).
  8. Place a cap on to the ultracentrifuge tube. Do not introduce any air bubbles and mix well.
  9. Load the other balanced buffer tube, place cap and avoid bubbles. Weigh two ultracentrifuge tubes. If there is over 0.05 g difference in weight, adjust the balanced tube with TE or CsCl2.
  10. Ultracentrifuge 35,000 rpm for 20 h at 4 °C. After that, you will see a viral particle band.
  11. In a laminar flow hood, carefully remove the tubes from the rotor, then open the cap.
  12. Using a 1 ml syringe with a 23 G1 needle, puncture the side of tube below the viral particle band (cloudy white).
  13. Aspirate the viral band (about 1 ml) carefully, avoid collecting other bands and impurities. If the viral band is over 1 ml, use another 1 ml syringe with 23 G1 needle and puncture at different position of tube to collect virus.
  14. Transfer virus directly to a Slide-A-Lyzer 10 K dialysis cassette that should be hydrated by immersing into dialysis buffer for 30 min.
  15. Insert the tip of the needle at a top corner of the cassette, and inject virus slowly.
  16. Transfer the virus cassette into an autoclaved 1 L baker containing 1,000 ml dialysis buffer with the lowest speed of stir bar spin.
  17. Dialyze at 4 °C for 2 h then change fresh buffer and dialyze overnight. On the second day, change 1,000 ml fresh buffer and dialyze for another 4 h.
  18. Carefully collect the virus by sucking with syringe to a clean sterilized tube, measure OD260 with 1:50 dilution.
  19. Calculate virus concentration (VP/ml): OD260 x 50 x 1010. Aliquot, store at -80 °C.

Recipes

  1. TE
    10 mM Tris-HCl (pH 8.0)
    1 mM EDTA
  2. Saturated CsCl2
    Add CsCl2 25 g to TE, and stir, repeat the addition until CsCl2 doesn’t dissolve anymore. Transfer the solution to 37 °C water bath, add more CsCl2. It takes about 100 g CsCl2 to saturate 50 ml buffer. Autoclave and store at RT.
  3. 5% deoxycholate
    5% deoxycholate in water, filter sterilized.
  4. 1% SDS (for washing the probe), filter sterilized.
  5. Balance buffer (10 ml)
    6.4 ml TE and 3.6 ml of saturated CsCl2
  6. Dializing buffer (4 L)
    10 mM Tris (pH 8.0)
    2 mM MgCl2
    5% sucrose

References

  1. Duffy, A. M., O'Doherty, A. M., O'Brien, T. and Strappe, P. M. (2005). Purification of adenovirus and adeno-associated virus: comparison of novel membrane-based technology to conventional techniques. Gene Ther 12 Suppl 1: S62-72.
  2. Graham, F. L. and Prevec, L. (1991). Manipulation of adenovirus vectors. Methods Mol Biol 7: 109-128.
  3. Guo, Z., Su, W., Ma, Z., Smith, G. M. and Gong, M. C. (2003). Ca2+-independent phospholipase A2 is required for agonist-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem 278(3): 1856-1863.

简介

腺病毒是有效的基因递送系统。 用于纯化腺病毒载体的标准方法基于使用氯化铯(CsCl)密度梯度与超速离心的组合。 该方法适合于小规模纯化,并且比柱层析或商业纯化试剂盒更便宜。

材料和试剂

  1. HEK293细胞
  2. 甘油
  3. 液氮
  4. 70%乙醇
  5. 蒸馏水
  6. SDS
  7. CaCl <2>
  8. MgCl 2
  9. CsCl 2(Thermo Fisher Scientific,目录号:BP1595-500)
  10. Tris-HCl(pH 8.0)
  11. TE
  12. EDTA
  13. SDS
  14. 蔗糖
  15. 5%脱氧胆酸盐(参见配方)
  16. 透析缓冲液(参见配方)
  17. 平衡缓冲区(参见配方)
  18. 饱和CsCl 2 (参见配方)

设备

  1. 离心机
  2. 超速离心机
  3. 层流罩
  4. 注射器
  5. 水浴
  6. Slide-A-Lyzer 10K透析盒(Thermo Fisher Scientific,目录号:66203)
  7. 光谱仪
  8. Ti50.2管

程序

  1. 在37℃下从液氮中解冻HEK293细胞悬浮液(使用在TE + 5%甘油中的腺病毒 20.5ml)。
  2. 加入1.23ml 5%脱氧胆酸盐至0.3%终浓度并涡旋
  3. 在冰上孵育30分钟(每10分钟涡旋)。 孵育期间,准备homoginizer:用70%乙醇洗涤,然后用蒸馏水,然后在紫外线下消毒。
  4. 均质化直至细胞悬浮液自由流动(设定为25次,三次,均化2分钟,每次冰上3分钟)。
    洗涤匀浆器:1×1%SDS,5×常规水,3×灭菌的dH 2 O,干燥。
  5. 孵育细胞悬浮在冰上15分钟(每5分钟涡旋)
  6. 在9000×g下旋转30分钟以在4℃下除去细胞碎片
  7. 将11.9ml饱和CaCl 2和20.5ml匀浆加载到超速离心管(适合Ti50.2管)上。
  8. 将盖子放在超速离心管上。 不要引入任何气泡并混匀。
  9. 装入另一个平衡缓冲管,放置盖子,避免气泡。 称重两个超速离心管。 如果重量差超过0.05g,用TE或CsCl <2>调节平衡管。
  10. 在4℃下超速离心35,000rpm 20小时。 之后,您将看到病毒颗粒带。
  11. 在层流罩中,小心地从转子上取下管,然后打开盖子
  12. 使用带有23G1针的1ml注射器,刺穿病毒颗粒带下面的管的侧面(混浊的白色)。
  13. 小心吸出病毒带(约1 ml),避免收集其他条带和杂质。 如果病毒带超过1 ml,使用另一个1毫升注射器与23 G1针和穿刺在不同位置的管收集病毒。
  14. 将病毒直接转移至Slide-A-Lyzer 10K透析盒,应通过浸入透析缓冲液30分钟使其水合。
  15. 将针尖插入录音带的顶角,慢慢注射病毒。
  16. 将病毒盒转移到含有1000ml透析缓冲液的高压灭菌1升面包机中,搅拌棒旋转速度最低。
  17. 在4℃透析2小时,然后更换新鲜缓冲液并透析过夜。 第二天,更换1000毫升新鲜缓冲液,再透析4小时。
  18. 通过用注射器抽吸至干净的灭菌管小心收集病毒,用1:50稀释度测量OD 260。
  19. 计算病毒浓度(VP/ml):OD <260> 50×10 10 。 等分,储存于-80℃

食谱

  1. TE
    10mM Tris-HCl(pH8.0) 1mM EDTA
  2. 饱和CsCl <2>
    向TE中加入CsCl 2 25g,搅拌,重复加入,直到CsCl 2不再溶解。 将溶液转移至37℃水浴,加入更多的CsCl 2。 其需要约100g CsCl 2以饱和50ml缓冲液。 高压灭菌并储存在室温。
  3. 5%脱氧胆酸盐 5%脱氧胆酸盐的水溶液,过滤灭菌
  4. 1%SDS(用于洗涤探针),过滤灭菌
  5. 平衡缓冲液(10ml)
    6.4ml TE和3.6ml饱和CsCl 2溶液
  6. 拨号缓冲区(4 L)
    10mM Tris(pH8.0) 2mM MgCl 2/
    5%蔗糖

参考文献

  1. Duffy,A.M.,O'Doherty,A.M.,O'Brien,T.and Strappe,P.M。(2005)。 腺病毒和腺相关病毒的纯化:小鼠的比较 基于膜的技术与常规技术。 Gene Ther 12 Suppl 1:S62-72。
  2. Graham,F.L.和Prevec,L。(1991)。 操纵腺病毒载体 方法Mol Biol 7:
  3. Guo,Z.,Su,W.,Ma,Z.,Smith,G.M.and Gong,M.C。(2003)。 Ca 2 + - 非依赖性磷脂酶A2是激动剂诱导的Ca 2 + 对血管平滑肌收缩的敏化。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pang, H. (2012). Purification of Adenovirus by Cesium Chloride Density Gradients. Bio-protocol Bio101: e201. DOI: 10.21769/BioProtoc.201;
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anita
medical university of Graz
what will happen if purified virus is stored in liquid nitrogen? will it affect the infection efficiency of virus ?
10/8/2013 3:57:58 AM Reply